RNA sequencing of astrocytes purified from adult brain cortices of TwKOastro mice (Knockout of Twnk in astrocytes generated with Gfap73.12:Cre driver, Jackson stock 012886)

To investigate astrocyte responses to mitochondrial dysfuncation, we induced a knockout of Twnk (encodes mitochondrial DNA helicase Twinkle) in mouse brain astrocytes (TwKOastro mice). This leads to depletion of mitochondrial genome copy number and its expression. Transcriptomes of 5 TwKOastro astrocyte preparations were compared to 6 control preparations. Astrocytes were purified from 3-3.5 months old control and TwKOastro mice using magnetic beads coated with ACSA-2 antibody (cortices from 3-4 mice per one preparation were used). RNA sequencing was done in the BGI Genomics, China. mRNA was enriched by Oligo dT selection, and reverse transcription was done using random N6 primer. End repair and adaptor ligation were followed by PCR amplification of the library. Quantification of transcript abundance was done using kallisto software, differential expression analysis was done using sleuth (PMID:27043002; PMID:28581496). TwKOastro mice = Gfap73.12Cre+;Twnkloxp/loxp or Gfap73.12Cre+;Twnkloxp/null. Control mice = Gfap73.12Cre-;Twnkloxp/loxp or Gfap73.12Cre-;Twnkloxp/null. TwKOastro mice are characterised in PMID:29302033. Generation of Twnkloxp/loxp mice is described in PMID:26924217. LoxP sites are located in the introns 1 and 3 of Twnk, Cre expression leads to a frameshift. Gfap73.12-Cre mice = JAX:012886.为探究星形胶质细胞（astrocyte）对线粒体功能异常的应答反应，我们在小鼠脑星形胶质细胞中敲除Twnk基因（该基因编码线粒体DNA解旋酶Twinkle），构建了TwKOastro小鼠模型。该操作会导致线粒体基因组拷贝数及其转录本丰度降低。我们对5份TwKOastro星形胶质细胞样本的转录组与6份对照样本的转录组进行了比较分析。 我们使用包被有ACSA-2抗体的磁珠，从3~3.5月龄的对照与TwKOastro小鼠脑中纯化星形胶质细胞（每份样本取自3~4只小鼠的大脑皮层）。转录组测序工作在中国华大基因（BGI Genomics）完成。通过寡聚dT磁珠富集法纯化mRNA，并使用随机六聚体引物进行反转录。在完成末端修复与接头连接后，对测序文库进行PCR扩增。使用kallisto软件对转录本丰度进行定量，使用sleuth软件完成差异表达分析（文献PMID：27043002；PMID：28581496）。 TwKOastro小鼠基因型为：Gfap73.12Cre+;Twnkloxp/loxp 或 Gfap73.12Cre+;Twnkloxp/null；对照小鼠基因型为：Gfap73.12Cre-;Twnkloxp/loxp 或 Gfap73.12Cre-;Twnkloxp/null。 TwKOastro小鼠的特征已在文献PMID：29302033中进行详细阐述。 Twnkloxp/loxp小鼠的构建方法详见文献PMID：26924217。 Twnk基因的第1和第3内含子区域插入了LoxP位点，Cre重组酶表达会导致该基因发生移码突变。 Gfap73.12-Cre小鼠的品系编号为JAX:012886。